人前列腺癌细胞骨转移潜能差异表达蛋白的研究

Differential proteomic analysis and function study of human prostate carcinoma cells with different osseous metastatic tendency

目的研究人前列腺癌不同骨转移潜能细胞株的差异表达蛋白质图谱，筛选前列腺癌骨转移相关蛋白并探讨其功能。方法应用蛋白质组学和免疫印迹等方法比较和筛选来源相同但具有不同骨转移潜能的前列腺癌细胞株（PC-3／骨转移亚克隆T3B／淋巴结转移亚克隆P2-4）的蛋白质表达差异。构建高迁移率族蛋白1（HMGB1）的真核表达载体Pgenesil-1／HMGB1siRNA转染至高骨转移潜能的T3B细胞中，通过动物实验观察HMGB1对前列腺癌细胞骨转移的影响。结果蛋白质组学技术获得6个有意义的蛋白质，分别参与细胞骨架构成、转录调控、磷酸化过程和物质代谢等功能。成功构建siRNA表达载体Pgenesil-1／HMGB1siRNA，转染后可使T3B细胞HMGB1的蛋白表达水平显著降低（P〈0．05）。动物实验显示，抑制HMGB1表达可明显降低T3B细胞的骨转移能力（P〈0．05）。结论高骨转移潜能的前列腺癌细胞株中存在与前列腺癌骨转移相关的蛋白质，HMGB1与前列腺癌骨转移密切相关。应用siRNA干扰技术能有效地抑制HMGB1基因的表达，同时也能有效抑制前列癌细胞的骨转移能力。

Objective To investigate the differential protein expression profiles of human prostatic carcinoma cells with different metastatic tendency and to screen the osseous metastasis associated proteins and investigate their function. Methods Proteomics and Western blotting were applied to screen and identify the differentially expressed proteins in the prostatic carcinoma cells of different lines： line T3B with high osseous metastasis potential, line P2-4 with high lymphatic metastasis potential, and their common parent cell line PC-3. The eukaryotic expression vector carrying human Pgenesil-1/HMGBlsiRNA was constructed and transfected into T3B cells by Lipofectamine 2000 and the positive clones was screened by G418. Pgenesil-1/HMGBlsiRNA/T3B, Pgenesil-1/T3B, and T3B cells were inoculated into the left ventricles of nude mice. Twelve weeks later the mice were killed. The number of osseous metastatic nodules and osseous metastasis inhibition rate were calculated. The mice metastatic tumor cells were identified by immunohistochemistry. Result Six differential expressed proteins, correlated with cystoskeleton, transcriptional control, cellular metabolism, and phosphorylation were identified by proteomics and Western blotting. The recombinant plasmid Pgenesil-1/HMGBlsiRNA was successfully constructed. The HMGB1 expression of the T3B cells transfected with Pgenesil-1/HMGBlsiRNA was significantly lower than those of the other 2 groups （both P 〈 0.05 ）. The number of osseous metastatic nodules of the mice inoculated with Pgenesil-1/HMGBlsiRNA/T3B was significantly less than those of the other 2 groups （both P 〈 0.05 ）. The metastatic osseous tumor cells were identified as the human prostatic carcinoma cells. Conclusion Osseous metastasis associated proteins exist in prostatic carcinoma cells of the hne with high osseous metastasis potential. HMGB1 is closely related to the osseous metastasis of human prostatic carcinoma cells, siRNA targeting HMGB1 specifically suppresses the expression of HMGB1 gene in the human prostatic carcinoma cells with high osseous metastasis potential and effectively inhibits the osseous metastasis.